POSITIVE RESULTS FINALLY

March 01^st.

      Today I made a gel by myself and Ms. Urban watched as I was putting it together to make sure I was doing everything right. I followed the Instructions on measuring the agarose, buffer and microwaving time in intervals then carefully adding Ethidium Bromide. Afterward I cross-linked the little dish to put the PCR reaction in while the gel was cooling. I then prepared the gel rig and poured water in it before setting the gel in. Afterwards I spun my PCR reaction from last week and added 1 microliter of the blue dye before putting it in the gel underwater in the gel rig.

      The process that was once almost impossible for me to accomplish in an hour took only a few minutes to do. I set the PCR reaction into the gel and turn the gel rig on then waited for 5 minutes to let the EtBr bind and the electricity flow through the gel.

      After five minutes I took the gel to the UV table to take a picture and see if the condition we set from last week worked. And the results were marvelous. The conditions worked perfectly, for the second group; with decreased water and MgCl₂. As shown below the bands we very clear and there wasn’t much smug in them.

KEY:

White bands= Positive results

Red-Orange smug= Dyes

Dark bands= original gel wells

   

As any lab experiment to verify that the conditions work and will work for the species we set up another experiment with the same conditions. The PCR reaction was small and using the same species. I left the reaction in the thermo cycler with the 51 ⁰ C annealing temp. In two weeks I will make another gel and see if it worked.

PCR REACTION IN ONE HOUR

Feb 23^rd

      Last experiment didn’t work very well with the species we used and/or the annealing temperature so this time we used three new species; ZAL 70, 71, 72. Mr. Adams and I reduced the water by a factor of 1.7 so that the concentration of the reaction remains the same. Instead of the regular 17.5 Microliters for a 25 Microliter reaction we made 14. 8. With this change we also increased the amount of MgCl₂ from 1.5 to 2.5 Microliters. In addition we made another row of regular water amounts so that we have a better idea of which conditions work best for the species.

      With practice, this time I put the two PCR reactions in an hour instead of the regular two. I am much faster with the lab equipment and putting together the experiment. I also know my way around fixing small mistakes such as Pipette usage or controlling amounts of Primers, Q-taq, dNTP’s, Buffer and water to get good results.

      With the remainder of my time in the lab I learned to Morphological way of identifying the species I work with in their molecular level. Mr. Adams introduced me to the shapes of Moths under the microscope and their morphology as a whole. The best way to identify the Species and place them in the right taxa is in fact through their reproductive system the shapes of the male genitalia helps place it in the right taxa; same thing with females.